Enzymatic crosslinking of dynamic hydrogels for in vitro cell culture

Abstract

Stiffening and softening of extracellular matrix (ECM) are critical processes governing many aspects of biological processes. The most common practice used to investigate these processes is seeding cells on two-dimensional (2D) surfaces of varying stiffness. In recent years, cell-laden three-dimensional (3D) scaffolds with controllable properties are also increasingly used. However, current 2D and 3D culture platforms do not permit spatiotemporal controls over material properties that could influence tissue processes. To address this issue, four-dimensional (4D) hydrogels (i.e., 3D materials permitting time-dependent control of matrix properties) are proposed to recapitulate dynamic changes of ECM properties. The goal of this thesis was to exploit orthogonal enzymatic reactions for on-demand stiffening and/or softening of cell-laden hydrogels. The first objective was to establish cytocompatible hydrogels permitting enzymatic crosslinking and stiffening using enzymes with orthogonal reactivity. Sortase A (SrtA) and mushroom tyrosinase (MT) were used sequentially to achieve initial gelation and on-demand stiffening. In addition, hydrogels permitting reversible stiffening through SrtA-mediated peptide ligation were established. Specifically, poly(ethylene glycol) (PEG)-peptide hydrogels were fabricated with peptide linkers containing pendent SrtA substrates. The hydrogels were stiffened through incubation with SrtA, whereas gel softening was achieved subsequently via addition of SrtA and soluble glycine substrate. The second objective was to investigate the role of dynamic matrix stiffening on pancreatic cancer cell survival, spheroid formation, and drug responsiveness. The crosslinking of PEG-peptide hydrogels was dynamically tuned to evaluate the effect of matrix stiffness on cell viability and function. Specifically, dynamic matrix stiffening inhibited cell proliferation and spheroid formation, while softening the cell-laden hydrogels led to significant increase in spheroid sizes. Matrix stiffness also altered the expression of chemoresistance markers and responsiveness of cancer cells to gemcitabine treatment. markers and responsiveness of cancer cells to gemcitabine treatment.